NOX enzymes are the primary source of cellular ROS under normal and pathological conditions. To date, 7 mammalian NOX family members have been described (NOX1-NOX5, DUOX1 and DUOX2). While NOX2 is mostly present in phagocytes, NOX1 is present in the colonic epithelium and DUOX2 is expressed in both the small and large intestine. Deficiencies in phagocyte-derived ROS due to defective NOX2 subunits (gp91phox, p47phox, p67phox, p22phox, p40phox) lead to chronic CGD, a primary immunodeficiency presenting with recurrent infections and an almost 50% rate of IBD. Single-nucleotide polymorphisms (SNPs) and rare hypomorphic variants in NOX2 subunits, although causing some attenuation in NOX2-derived ROS, lead to adult and very early onset IBD without causing CGD. Moreover, inactivating missense variants in NOX1 and DUOX2 have also been associated with very early onset IBD. Meanwhile, studies in mice have shown that NOX1-derived ROS, through N-formyl peptide receptor signaling or stimulation by symbiotic lactobacilli, can drive colonic epithelial repair. Together, these findings support our goal of defining the role of NOX complex-derived ROS in IBD and intestinal barrier homeostasis. To do this, we have focused on the study of CGD-associated IBD (CGD IBD) in order to first determine the role of phagocyte (NOX2)-derived ROS in IBD pathogenesis. We have developed a translational research model including targeted in vitro and murine studies, and the study of contextualized human specimens while developing, in parallel, a clinical treatment protocol for patients with CGD IBD. Our studies in CGD mice have shown that although these mice do not spontaneously develop colitis, there are strain-specific patterns of colitis susceptibility, which are largely mediated by the intestinal microbiome established at birth. These findings have supported further investigation of the intestinal microbiome in patients with CGD, including patients with and without colitis, before and after hematopoietic stem cell transplantation. Our ongoing intestinal microbiome studies in patients with CGD have already helped identify surrogate markers of intestinal inflammation, as well as significant variables affecting the CGD microbiome signature in the presence or absence of IBD. In addition to the study of the intestinal microbiome, our multifaceted approach to the study of CGD IBD includes the investigation of the immunological role of intestinal hematopoietic and non-hematopoeitic cells in relation to cell-type specific NOX complex function and ROS production. Although currently being established in in vitro and murine models, our experimental methods will be optimized for the study of human specimens through an investigational review board (IRB)-approved clinical protocol.